Downstream processing of viral vectors and vaccines.

Viral vectors and viral vaccines more and more play an important role in current medical approaches. Gene vectors like adenoviruses, adeno-associated viruses or retroviruses are the vehicles being developed for delivering genetic material to the target cell in gene therapy. Viral vaccines, like attenuated or inactivated rabies virus, influenza virus or hepatitis virus vaccines, are powerful tools to limit the number of serious viral infections and pandemics. Higher safety demands, that is, reduction of side effects, by regulatory authorities like Food and Drug Administration (FDA) and European Agency for the Evaluation of Medicinal Products (EMEA), nowadays force developers as well as manufacturers to improve their production and purification processes for viral vectors and vaccines. Like for influenza viral vaccines, manufacturers begin to switch from egg cultivation to mammalian cell culture systems. Also within the purification procedure, a clear trend from classical purification methods like sucrose gradient centrifugation towards more sophisticated techniques like tangential flow filtration and liquid chromatography can be observed.

The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition.

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.

Structure-based design of a potent chimeric thrombin inhibitor.

Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (Ki) in the 10 nM concentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (Ki in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.

Structure of leech derived tryptase inhibitor (LDTI-C) in solution.

The three-dimensional solution structure of the leech derived tryptase inhibitor form C (LDTI-C), an inhibitor of 46 amino acids which contains 3 disulfide bridges, has been determined using 2D NMR spectroscopy. The 3D structure was determined on the basis of 262 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 25 phi angles, supplemented by 3 psi and 15 chi 1 angles. The core of LDTI-C is very well defined and consists of a short 3(10)-helix-loop and a short two-stranded antiparallel beta-sheet between residues 13-14 and 20-21. The N-terminus is fixed to the core by two disulfide bridges, while the C-terminus is connected to the beta-sheet via the third disulfide bridge. The binding loop in LDTI exhibits lowest energy conformations belonging to the canonical conformation of serine proteinase inhibitors.

Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.

A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.